Adenoviruses have become important tools in vaccine development and gene transfer as vectors for in vitro, in vivo and ex vivo transfer of heterologous, therapeutic and/or immunogenic genes. Adenoviruses offer several advantages compared to other vectors; for example, they can be produced at high titers and can infect resting and nondividing cells. Furthermore, the adenoviral genome can be manipulated to accommodate foreign genes of up to about 8 kb in length. In addition, as an adenoviral vector does not insert its DNA into the chromosome of a cell, its effect is impermanent and unlikely to interfere with the cell's normal function. Lastly, live adenoviruses have been safely used as human vaccines (Horwitz, “Adenoviruses,” in Virology (Fields et al., eds, Lippincott-Raven Publishers, Philadelphia, 3rd ed., pp. 2149-2171, 1996; Berkner et al., J. Virol., 61, 1213-1220 (1987); Couch et al., Amer. Rev. Respir. Dis. 88:394-403, 1963: Franklin et al., J. Infect. Dis. 124:148-154, 1971; and Franklin et al., J. Infect. Dis. 124:155-160, 1971). Consequently, adenovirus vectors are often used to introduce a gene (or genes) of interest into a host cell.
In replication-defective Ad vectors, the E1 genes, which are required for Ad replication, are deleted. The transgene expression depends on a foreign promoter (e.g., Wilkinson & Akrigg, Nucl. Acids Res. 20:2233-2239, 1992; Massie, et al. J. Virol. 72:2289-2296, 1998). The most commonly used replication-defective Ad vector is derived from adenovirus type 5.
Unlike replication-defective Ad vector, E1 genes are maintained in replication-competent Ad vectors. In some replication competent Ad virus vectors, the viral E3 genes are deleted and a transgene is inserted downstream about 80 map unit (mu) of the adenovirus genome. The expression of the transgene is under the control of adenoviral major late promoter (MLP).
A number of gene transfer studies have employed replication-defective adenovirus vectors that have heterologous promoters to express a transgene. However, the prior art has provided conflicting reports regarding the need for a heterologous promoter for replication competent adenovirus vectors. It has been reported that the gene encoding hepatitis B virus surface antigen (HBsAg) was easily transcribed when it was incorporated into a replication competent adenovirus vector under the control of its own promoter or a heterologous promoter. However, the HBsAg mRNA was often poorly translated (e.g., Tiollais et al., Science 213:406, 1981; Sait et al., J. Virol. 54:711, 1985) and did not achieve high level expression of the protein. During the same time period, positive results were also reported for expressing the HBsAg gene without a foreign promoter (Morin, et al., Proc. Nat. Acad. Sci. USA, 84:4626, 1987). Thus, there was no suggestion or teaching in the art that it would be of benefit to incorporate a heterologous promoter into a replication-competent adenovirus to regulate expression of a transgene.
The current invention provides a method for achieving high level expression of a transgene using replication-competent adenovirus vectors containing a hybrid gene regulation unit. The hybrid gene regulation unit comprises a CMV promoter and an adenovirus tripartite leader sequence. These components have been incorporated into replication-defective adenoviruses to regulate transgene expression (e.g., Wilkinson & Akrigg, Nucl. Acids Res. 20:2233-2239, 1992; Massie, et al. J. Virol. 72:2289-2296, 1998). However, before the present invention, replication competent vectors comprising both the CMV and tripartite sequences were not known in the art. The present invention thus provides a new series of replication competent adenovirus vectors that comprise a hybrid regulatory unit, which provide high levels of gene expression relative to previous vectors.
In particular embodiments, the invention also provides replication competent adenovirus vectors containing a hybrid gene regulation unit that controls expression of one or more sequences encoding an HIV polypeptide. Recent advances in developing a vaccine against HIV/AIDS has led to the realization that multi-component vaccines eliciting both humoral and cellular immune responses are often important for achieving a successful vaccine. Evidence from developmental HIV and simian immunodeficiency virus (SIV) vaccine studies indicates that vaccines encoding multiple viral antigens can induce broader immune responses and/or greater protective efficacy against viral infection (e.g., Ourmanov et al., J Virol 74:2960-65, 2000; Kong et al., J Virol 77:12764-72, 2003; Hel et al., J Immunol 169:4778-87, 2002; Zhao et al., J Virol 77:8354-65, 2003; Patterson et al., J Virol 78:2212-21, 2004; Negri et al., J Gen Virol 85:1191-201, 2004; Mossman, et al., AIDS Res Hum Retroviruses 20:425-34, 2004.) In addition to the viral structural and enzymatic proteins, Env, Gag and Pol, viral regulatory and accessory proteins are important potential vaccine components (e.g., Robert-Guroff M. DNA Cell Biol 21:597-98, 2002). Tat, Rev, and Nef, in particular, have been targeted by several laboratories for vaccine development (Negri et al., Mossman et al, both supra; Osterhaus, et al., Vaccine 17:2713-4, 1999; Cafaro et al., Nat Med 5:643-50, 1999; Muthumani et al., J Med Primatol 31:179-85, 2002; Verrier, et al. DNA Cell Biol 21:653-8, 2002; Richardson et al., DNA Cell Biol 21:637-51, 2002; Patterson et al., DNA Cell Biol 21:627-35, 2002; Tikhonov et al., J. Virol. 77:3157-66, 2003; Makitalo et al., J Gen Virol. 85:2407-19, 2004).
HIV-1 Tat is a small nuclear protein that has a variety of activities. In addition to functioning as a transcriptional transactivator of HIV gene expression, Tat is released from productively infected cells and can be taken up and imported into the nucleus of many different cell types. There, it promotes HIV replication or modulates the expression of cellular genes (transcription factors, cytokines, and genes that regulate the cell cycle and are important for HIV replication) (see, e.g., Barillari, et al., Clin Microbiol Rev 15:310-26, 2002; Caputo, et al., Curr HIV Res, 2004; for reviews).
Because it is produced early in the HIV replication cycle, vaccine induced immune responses to Tat might inhibit Tat functions, abrogating both HIV transactivation and the deleterious effects of Tat on uninfected, bystander cells. However, the potential use of Tat as a vaccine candidate is controversial. On the positive side, studies of HIV-infected patients and SIV-infected non-human primates suggest that an immune response to Tat has a protective role and may control the progression of the disease. Anti-Tat antibody responses and CTLs have been associated with non-progression to AIDS in infected individuals (e.g., van Baalen et al., J Gen Virol 78:1913-8, 1997; Re, et al., J Clin Virol 21:81-9, 2001; Reiss, et al., J Med Virol 30:163-8, 1990; Zagury, et al., J Hum Virol 1:282-92, 1998). Furthermore, a study in macaques infected with SIV demonstrated that anti-Tat CTLs are important to control of early virus replication (Allen, et al., Nature 407:386-90, 2000). As a vaccine candidate, however, mixed results have been obtained using different approaches. Immunization with active Tat protein has led to strong protection of cynomolgus macaques in pre-clinical vaccine studies (Cafaro, et al., Nat Med 5:643-50, 1999). In contrast, immunization with inactivated or native Tat has shown minimal or no protection of rhesus macaques (Pauza, et al., Proc Natl Acad Sci USA 97:3515-9, 2000; Silvera, et al., J Virol 76:3800-9, 2002). Thus, there is a need in the art to define the role of Tat in HIV vaccines. This invention addresses that need.
HIV Nef protein is also an attractive component of an HIV vaccine. The Nef protein is required for high-titer HIV replication in vivo and critically important for AIDS development. Nef is expressed early in the viral life cycle from the pre-integrated viral genome (Wu & Marsh, Science 293:1503-1506, 2001), rendering it suitable for early targeting by cellular immune responses. Nef is expressed on the surface of infected cells (Fujii, et al., Vaccine 11:1240-1246, 1993), and has been implicated as a target of antibody-dependent cellular cytotoxicity (Yamada, et al., J. Immunol. 172:2401-2406, 2004), suggesting that a vaccine elicited antibody response to Nef could also lead to rapid killing of HIV-infected cells expressing Nef.
Nef-mediated down-regulation of both CD4 (Garcia & Miller Nature 350: 508-511, 1991) and MHC class I (Schwartz, et al. Nat. Med. 2:338-342, 1996) molecules from the surface of infected cells has been well documented. These effects may affect the immunogenicity of nef-encoded vaccine candidates by diminishing the expression of Nef-peptide-MHC-I complexes on the cell surface as targets for induction of CTL responses, and by diminishing CD4 help, necessary for elicitation of both B and T-cell responses and induction of cellular memory.
The N-terminal myristoylation site of Nef is required for its association with the cell membrane. It was previously reported in in vitro studies showing that deletion of the myristoylation site prevented down modulation of both CD4 and MHC-I molecules since Nef was no longer anchored at the cell membrane (Peng & Robert-Guroff, Immunol. Lett. 78:195-200, 2001). At the same time, although other regions of Nef have been shown to participate in the modulation of CD4 and MHC-1 cell surface expression and their deletion might have similarly abrogated the modulatory functions, the simple N-terminal deletion preserved almost all known Nef B- and T-cell epitopes maintaining the suitability of the mutated Nef as a vaccine candidate.
There is also a need to address the role of Nef or modified Nef forms in a vaccine. This invention provides an expression system that results in high level expression of the products of a transgene, nef. Accordingly, the invention also addresses this need.